The invention relates generally to transgenic nonhuman mammals producing procollagen or collagen in their milk.
Collagen is a family of fibrous proteins present in all multicellular organisms. Collagen forms insoluble fibers having a high tensile strength. Collagen is the major fibrous element of skin, bone tendon cartilage, blood vessels and teeth. It is present in nearly all organs and serves to hold cells together in discrete units. Recently, collagen has assumed a therapeutic importance in reconstructive and cosmetic surgical procedures.
The process by which collagen is expressed, processed and ultimately assembled into mature collagen fibers is complex. At least 28 distinct collagen genes have been reported, whose expression products combine to form at least 14 different forms of collagen. Different forms of collagen are associated with different tissue types. For example, type I collagen is distributed predominantly in skin, tendon, bone and cornea; type II collagen in cartilage, invertebrate discs and vitreous bodies; type III collagen in fetal skin, the cardiovascular system and reticular fibers; type IV collagen in basement membranes; and type V collagen in the placenta and skin. Collagen types I, II and III are the most abundant forms and have a similar fibrillar structure. Type IV does not exist in fibrils but rather forms a two-dimensional reticulum constituting the principal component of the basal lamina.
A collagen gene is expressed to give a polypeptide termed a procollagen linked at its N-terminal to a signal peptide. The procollagen polypeptide contains a central segment that is ultimately found in mature collagen between N- and C-terminal propeptides. For procollagen xcex11(I), the procollagen polypeptide is about 160 kDa, the mature collagen polypeptide about 90 kDa and the propeptides about 45 kDa. The signal peptide is linked to the amino end of the N-terminal propeptide. The amino acid composition of propeptides differs from the mature peptide. The mature peptide has an unusual repeating structure in which glycine occurs as nearly every third amino acid and there is a high proportion of proline residues. The propeptides have a role in promoting interchain assembly of procollagen chains into triplex structures.
Following expression of signal peptide-procollagen polypeptides, a series of posttranslation modifications occur in the course of assembly and secretion of procollagen. In fibroblasts, the following modifications have been identified: cleavage of signal peptides at the N-termini of the chains; hydroxylation of the Y-position proline and lysine residues, hydroxylation of a few X-position proline residues; addition of galactose or galactose and then glucose to some of the hydroxylysines, addition of a mannose-rich oligosaccharide to the C propeptides, association of the C-terminal propeptides through a process directed by a structure of these domains, formation of both intra and interchain disulfide bonds in the propeptides. Following these modifications, the procollagen chains assemble into a trimeric helix composed of three procollagen chains. In synthesis of some forms of collagen, the three procollagen chains are of the same type; in synthesis of other forms of collagen, the three procollagen chain are heterologous. For example, type I collagen contains two xcex11(I) chains and one xcex12(I) chain. Individual chains assemble into trimers by interactions of propeptides. These interactions include formation of both intrachain and interchain disulfide bonds in the propeptides.
On completion of processing and assembly, procollagen trimers are secreted from the cell and subject to further extracellular modifications. The N- and C-terminal propeptides are cleaved from the mature collagen peptide by specialized enzymes termed procollagen N-proteinase and procollagen C-proteinase. The cleavage reaction releases individual trimers of mature collagen having a molecular weight of about 285 kDa (termed tropocollagen). Individual trimers spontaneously assemble into higher order structures. These structures are then solidified by lysyl oxidase conversion of some lysine and hydroxylysine residues to aldehyde derivatives that form interchain crosslinks. The final product constitutes high molecular weight insoluble fibrils that can fulfill the natural and surgical structural roles noted above. In all, the modification process requires at least eight specific enzymes, and several nonspecific enzymes, and requires modification of over one hundred amino acids. See Prockop et al., New England J. Med. 311, 376-386 (1984) (incorporated by reference in its entirety for all purposes).
The utility of collagen in surgical processes has led to attempts to express recombinant collagen genes as a source of collagen. For example, a genomic DNA segment encoding human cartilage procollagen xcex11(II) and a minigene version thereof (lacking most internal intronic sequences) have been expressed in 3T3 mouse fibroblast, a cell line producing endogenous collagen type I. See Ala-Kokko et al., J. Biol. Chem . 266, 14175-14178 (1991); Olsen et al., J. Biol. Chem. 266, 1117-11121 (1991)) (each of which is incorporated by reference in its entirety for all purposes). A cDNA encoding procollagen xcex12(V) has been expressed in mouse fibroblasts expressing endogenous proxcex11(V). See Greenspan, Proc. Natl. Acad. Sci. USA 84, 8869-8873 (1987)) (incorporated by reference in its entirety for all purposes). Heterotrimers were deposited predominantly in the extracellular matrix of the cell layer. A cDNA encoding the human proxcex11(I) chain has been expressed in a human fibrosarcoma cell line producing endogenous collagen type IV. See Geddis and Prockop, Matrix 13, 399-405 (1993) (incorporated by reference in its entirety for all purposes). About two percent of transformed cell lines secreted homotrimeric proxcex11(I) chains. These chains were overmodified compared with normal proxcex11(I) chains as judged by SDS PAGE analysis. Transgenic mice exhibiting systemic expression of mutated forms of procollagen genes have also been reported. See Stacey et al., Nature (1988) 322, 131-136; Khillan et al., J. Biol. Chem. 266, 23373-23379 (1991); WO 92/22333. Most such mice were born dead or severely deformed.
Mammalian cellular expression systems are not entirely satisfactory for production of recombinant proteins because of the expense of propagation and maintenance of such cells. An alternative approach to production of recombinant proteins has been proposed by DeBoer et al., WO 91/08216, whereby recombinant proteins are produced in the milk of a transgenic animal. This approach avoids the expense of maintaining mammalian cell cultures and also simplifies purification of recombinant proteins.
Although the feasibility of expressing several recombinant proteins in the milk of transgenic animals has been demonstrated, it was unpredictable whether this technology could be extended to the expression of an multimeric protein requiring extensive posttranslational modification and assembly, such as collagen. Because mammary gland cells naturally produce only low levels of endogenous collagen type IV (David et al., Expl. Cell. Res. 170, 402-416 (1987)), it was uncertain whether these cells possessed the necessary complement and activity of enzymes for proper modification, assembly and secretion of other types of collagen, particularly, at high expression levels. If not properly modified, collagen might accumulate intracellularly rather than being secreted. Moreover, the large size of trimeric procollagen ( greater than 420 kDa) in comparison with other milk protein might have been expected to clog the secretory apparatus. The health and even viability of transgenic animals expressing exogenous collagen in their mammary glands was also uncertain. Inappropriate accumulation of collagen in the mammary gland might have impaired mammary gland development and resulted in cessation of lactation. Even low levels of secondary expression in tissues other than the mammary gland could have resulted in lethal accumulation of collagen deposits.
Notwithstanding the above uncertainties and difficulties, the invention provides inter alia healthy transgenic mammals secreting procollagen or collagen into their milk.
The invention provides transgenic nonhuman mammals useful for production of procollagen or collagen. The mammals have a transgene comprising a mammary-gland specific promoter, a mammary-gland specific enhancer; a secretory DNA segment encoding a signal peptide functional in mammary secretory cells of the transgenic mammal, and a recombinant DNA segment encoding an exogenous procollagen polypeptide. The recombinant DNA segment is operably linked to the secretory DNA segment to form a secretory-recombinant DNA segment which is, in turn, operably linked to the promoter and enhancer. In adult form, the nonhuman mammal bearing the transgene, or a female descendant of the mammal, is capable of expressing the secretory-recombinant DNA segment in the mammary secretory cells to produce a form of the exogenous procollagen polypeptide that is processed and secreted by the mammary secretory cells into milk as exogenous procollagen or collagen. Usually, the exogenous procollagen or collagen is secreted in trimeric form. The concentration of procollagen or collagen in the milk is usually about 100 xcexc/ml and sometimes 1 mg/ml or more. The exogenous procollagen or collagen polypeptide is usually human, e.g., proxcex11(I). The recombinant DNA segment can be cDNA, genomic or a hybrid. In some genomic DNA segments, a segment of the first intron is deleted to remove regulatory sequences. Some transgenic nonhuman mammals have a first transgene encoding a proxcex11(I) polypeptide and a second transgene encoding a proxcex12(I) polypeptide. The two transgenes are capable of being expressed to produce forms of xcex11(I) and xcex12(I) procollagen that are processed and secreted by the mammary secretory cells into milk as a trimer comprising at least one chain of xcex11(I) procollagen or collagen and at least one chain of xcex12(I) procollagen or collagen. Preferred species of transgenic mammals include bovine and murine.
In another aspect, the invention provides milk from transgenic nonhuman mammals as described above. The milk comprises procollagen or collagen.
The invention further provides transgenes for expressing procollagen or collagen. One such transgene comprises a casein promoter, a casein enhancer, a cDNA segment encoding a procollagen signal segment linked in-frame to a procollagen xcex11(I) polypeptide, and a 3xe2x80x2 flanking DNA segment from a gene encoding the procollagen polypeptide. The cDNA segment is operably linked at its 5xe2x80x2 end to the promoter and the enhancer, and at its 3xe2x80x2 end to the 3xe2x80x2 flanking segment. Another transgene comprises a casein promoter, a casein enhancer and a genomic DNA segment comprising a segment from a 5xe2x80x2 untranslated region to a 3xe2x80x2 flanking region of a procollagen xcex11(I) gene, operably linked to the promoter and the enhancer.
In a further aspect, the invention provides a stable mammary gland cell line having a transgene. The transgene comprises a mammary-gland specific promoter, a mammary-gland specific enhancer, a secretory DNA segment encoding a signal peptide functional in the cell line, and a recombinant DNA segment encoding an exogenous procollagen polypeptide operably linked to the secretory DNA segment to form a secretory-recombinant DNA segment, the secretory-recombinant DNA segment being operably linked to the promoter and to the enhancer. The cell line can be induced by a lactogenic hormone to express the transgene to produce a form of the exogenous procollagen polypeptide that is processed and secreted by the cell lines as exogenous procollagen or collagen in trimeric form.